Vol. 128, No. 2 Printed in U.S.A.

JOURNAL OF BACTERIOLOGY, Nov. 1976, P. 604-608 Copyright © 1976 American Society for Microbiology

tolF Locus in Escherichia coli: Chromosomal Location and Relationship to Loci cmlB and tolD JOHN FOULDS

National Institute of Arthritis, Metabolism and Digestive Diseases, Bethesda, Maryland 20014 Received for publication 3 May 1976

The tentative map position on the Escherichia coli chromosome of the tolF locus, determining tolerance to colicins A, E2, E3, K, and L, has been confirmed by three-point transduction. It lies between the aroA and pyrD loci at about 21 min on the linkage map of Bachmann et al. (1976). The cmlB locus, determining increased resistance to the antibiotics chloramphenicol and tetracycline, also lies in this region (Reeve, 1966). Phenotypic and genetic comparison of isogenic strains that carry a mutation in either the tolF or cmlB locus makes it likely that these loci are closely related or identical. The tolD locus determining tolerance to colicins E2 and E3 as well as increased resistance to antibiotics has been reported to be located close to the aroA locus as a result of conjugation experiments (Eriksson-Grennberg et al., 1965). However, tolD did not cotransduce with any of several loci in this region, indicating that the mutation is not located within the region of the genetic map corresponding to approximately 19 to 22.5 min.

Serratia marcescens strain JF246 produces a bacteriocin (8) that is apparently closely related to colicin L produced by Escherichia coli strain 398. Antibody prepared against pure bacteriocin JF246 inactivated colicin L-398. In addition, mutants selected as tolerant to bacteriocin JF246 (10) were also tolerant to colicin L-398 and vice versa. Thus, following the suggestion of Fredericq (11) and P. Reeves (personal communication), I will refer to this material as colicin L-JF246. tolF mutants were selected as tolerant to colicin L-JF246 (10), whereas cmlB mutants were selected as resistant to chloramphenicol (14). tolF mutants were shown to have altered sensitivities to a variety of colicins, dyes, and detergents (10). cmlB mutants were shown to have increased resistance to chloramphenicol and tetracycline (14). This paper demonstrates that isogenic strains carrying tolF or cmlB have identical phenotypic responses to a variety of colicins, bacteriophages, dyes, detergents, and antibiotics. Genetic mapping and complementation tests confirm this identity. Until the nature of the tolF and cmlB gene product is clarified, I propose that this locus be called tolF. Tolerance to colicins is a more striking phenotypic effect than increased resistance to antibiotics. The colicin tolerance in some cases extends over a 4,000-fold concentration range, whereas the increase in resistance to antibiotics extends over a two- to threefold

A tolF strain was compared with a tolD strain for two reasons. First, the tolD strain was selected as a spontaneous mutant resistant to an antibiotic (ampicillin) (7) and was later characterized as tolerant to colicins E2 and E3 and somewhat resistant to chloramphenicol (2). Second, the tolD mutation was located by conjugation experiments on the genetic map near the tolF locus (2). I have demonstrated that the tolF can be cotransduced with pdxC, aroA, pyrD, and fabA, whereas tolD does not cotransduce with any of these loci. Thus the tolF and tolD loci are distinct. MATERIALS AND METHODS Microorganisms and media. E. coli K-12 strains used for these studies are described in Table 1. The medium has been previously described (10). Normally, cells were grown in L broth containing 2.5 mM CaCl2 or minimal medium supplemented with 0.2% glucose, streptomycin sulfate (100 ,ug/ml), and appropriate growth supplements. These media were solidified with 1.5% agar or 0.7% agar (soft agar). Bacteriophage Pivir was obtained from John Cronan, and phage f2 was obtained from Peter Model. Genetic procedures and strain construction. Before a conjugation experiment, the mating ability of the F' donor culture was confirmed first by demonstrating sensitivity of the culture to phage f2 and second by demonstrating the ability of the culture to donate appropriate genetic markers to a recA recipient. Growth of parental strains was generally inhibited by streptomycin or by the omission of re-

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VOL. 128, 1976

THE tolF LOCUS

TABLE 1. Escherichia coli K-12 strains Strain

AB2856 AT3143

CY99

Gllel

Reference or

Genotypea

source

aroA357 ilv-7 arg-3 thi-1 pro-2 his-4 pdxC3 pyrF ilv-277 metB65 his-53 purE41 proC24 cyc-1 xyl-14 lacY29 str-77 tsx-63 pyrD34 thr-1 his-i trp-1 galG xyl-7 mtl-2 recAl str-118 F '106 pyrD+ ilv metB tolD

JF557 JF558 JF560 JF561 JF562 JF568

(HfrC) thyA tolFf (HfrH) thyA tolFll (HfrH) KL185, aroA357 thyA b JF556, aroA + tolF4 JF556, aroA + cmlBb RE110 recAb JF557, recA tolF4b JF558, recA cmlBb AT3143, pyrF+ pdx+

KL16-99

recA thi ser-1l drm-3

JF404-4a JF404-lla JF556

(14)

A. L. Taylor

J. Cronan

(2) (10) (10)

b

aroA357b

B. Low

relA

KL185

thi-1 pyrD34 his-68 B. Bachmann trp-45 mtl -2 xyl-1 7, galK35 str-118 Xr, ARE107 proA trp his lacY (14) ton cmlB strA RE110 pyrD galE (16) UC1098 fabA2 strA (4) a Genetic nomenclature of Bachmann et al. is used (1). b Unless otherwise specified, all other markers are the same as in the indicated parental strain. c Preparation of strain described in this paper. quired supplements in the plating medium to allow development and identification of merodiploid exconjugants. Growth of phage Plvir and transduction procedures were as described by Signer (19). Direct selection of tolF or cmlB transductants was not successful, presumably due to a long lag before expression of the phenotype. This may reflect a requirement for complete removal of a component of the wild-type cell envelope before a transductant is phenotypically tolerant to colicin. Construction of isogenic strains that carried either tolF or cmlB mutations was accomplished by first introducing aroA357 into strain KL185. Phage Plvir, grown on strain JF568, was used to infect strain KL185, and after removal of unabsorbed phage, the aroA357 transductants were enriched, isolated, and purified by using the penicillin enrichment technique described by Rossi and Berg (17). A thyA mutant was isolated from this aroA pyrD

605

strain by using trimethoprim (20), and the resulting strain was labeled JF556. Next, phage Plvir lysates prepared on either strain JF404-4a or strain RE107 were used to introduce aroA+ tolF or aroA+ cmlB, respectively, into strain JF556 by transduction. These transductants were labeled JF557 and JF558 (Table 1). The recA derivative of strains JF557 and JF558 was prepared by conjugation with strain KL16-99 previously described (10), except that recA recombinants were picked, purified, and characterized from among a population of thyA + recombinants. The recA strains were labeled JF561 and JF562. Strain JF560 was prepared from a thyA derivative of strain RE110 by the same procedure. Sensitivity to colicins was determined by the plate assay previously described (10). Sensitivity to detergents, dyes, and ethylenediaminetetraacetic acid was determined as described by Davies and Reeves (5). Sensitivity to antibiotics was determined by using Sensi-Discs obtained from BBL, Cockeysville, Md. Up to 12 discs were placed on a single LC agar plate that had been overlaid with 2.5 ml of LC soft agar containing about 107 cells. After overnight incubation, sensitivity was scored by measuring a distinct zone of inhibition of growth of the test strain, whereas resistance was expressed as no detectable inhibition or a markedly decreased (at least 3 mm in diameter) size of zone of inhibition.

RESULTS Position of tolF and cmlB loci on the genetic map. Two independently isolated spontaneous tolF mutants in strain HfrH(JF404) were selected for transduction studies. For most studies, however, only the allele in strain JF404-4a was used. Table 2 describes the linkage of tolF and cmlB to several loci located between 20 and 21.5 min on the genetic map of Bachmann et al. (1). Clearly, tolF and cmlB share similar linkage relationships with pyrD and pdxC. (Compare Table 2, crosses 1, 2, 3, 5, and 6 with crosses 7 and 8). The three-factor transduction experiments summarized in Table 3 position both the tolF and cmlB loci between aroA and pyrD. The data in Table 4 report my attempts to separate tolF and cmlB loci by transduction. The mutations in these strains are closely linked but not identical. Two tolF+ recombinants were observed among 1,750 pyrD+ transductants. pyrD+ recombinants were also selected after conjugation between HfrH strain JF404-4a (pyrD+ toiF) and strain JF558 (pyrD cmlB). All of 800 pyrD+ recombinants tested were resistant to colicin A (tolF or cmlB). The low frequency of recombination between the tolF and cmlB strains indicates that the two mutations are located close to one another on the chromosome and may affect the same gene. The position of tolF on the genetic map is presented in Fig. 1, which summarizes the results in Tables 2 and 3.

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J. BACTERIOL.

FOULDS TABLE 2. Cotransduction of tolF and cmlB loci with loci close to pyrD locus Bacterial strains and relevant markers

Cross no.

Recipient

Donor

tolF locus 1

Selected marker

Transductants with unselected donor marker/total transductants tested

Cotransduction frequency

(%)

AT3143 (pdxC)

JF557 (pyrD tolF4)

pyrD+

151/250 tolFNa

2

JF557 (pyrD tolF4)

AT3143 (pdxC)

pdxC+

134/250 tolF

3

UC1098 (fabA)

JF557 (pyrD tolF4)

pyrD+

116/200 tolF+

4

JF557 (pyrD tolF4)

UF1098 (fabA)

fabA +

120/200 fabA 91/250 toIF

JF404-4a (tolF4) JF404-lla (tolFll)

JF544 (pyrD) JF544 (pyrD)

pyrD + pyrD +

125/250 pyrD 53/100 tolF 61/100 tolF

60 9.2 54 6.8 58 60 36 50 53 61

KL185 (pyrD) AT3143 (pdxC) AB2856 (aroA)

pyrD+ pdxC+ aroA +

145/250 cmlB 141/250 cmlB 135/250 cmlB

58 56 54

5 6 cmlB locus 7 8 9

RE107 (cmlB) RE107 (cmlB) RE107 (cmlB) Unselected marker.

a

23/250Opdx 17/250 pyrD

TABLE 3. Reciprocal three-factor cotransductions of tolF or cmlB with aroA and pyrD

1

Bacterial strains and relevant markers Donor Recipient AB2856 (aroA) JF557 (pyrD toiF)

2

JF557 (pyrD toiF)

3 4

Cross

no.

Selected No. semarker .

lected~~.

Distribution of unselected markers

pyrD+

250

aroA + tolF+ aroA + tolF aroA tolF+ aroA tolF 4 112 26 108

AB2856 (aroA)

aroA +

250

pyrD+ tolF+ pyrD+ tolF pyrD tolF+ pyrD tolF

AB2856 (aroA)

JF562 (pyrD cmlB)

pyrD +

250

JF562 (pyrD cmlB)

AB2856 (aroA)

aroA +

250

130 102 3 15 aroA + cmlB + aroA + cmlB aroA cmlB + aroA cmlB 111 2 119 18 pyrD + cmlB + pyrD + cmlB pyrD cmlB + pyrD cmlB 4 17 104 125

TABLE 4. Linkage between cmlB and tolF Selected marker e

No. N of expt

Transductants with wild type phenotype/total transductants tested

pyrD+ pyrD+

3 2

1/950 1/800

Bacterial strains

Donor.Recie Recipient

Donor

RE107 (cmlB) JF404-4a (tolF4)

JF557 (pyrD tolF) JF558 (pyrD cmlB)

The tolD locus is apparently unlinked by transduction to any of the loci in this figure, for although the tolD locus could be transferred by conjugation (2; unpublished observations), linkage by transduction between this locus and any of the loci studied was not observed. I found less than 0.4% cotransduction of tolD with pdxC, aroA, pyrD, or fabA. Phenotype of tolF and cmlB strains. Beginning with strain JF556 (aroA pyrD), I constructed nearly isogenic strains by transduction. These strains differed only in that strain JF557 carried aroA + tolF from strain JF404-4a and strain JF558 carried the aroA+ cmlB from strain RE107. The sensitivity of the parental strain and the two transductants to a variety of agents is shown in Table 5. The phenotypes of

the tolF and cmlB strains were identical. Complementation of tolF and cmlB by F'106. To prevent recombination between the chromosome and the episome, recA derivatives of strains JF557 and JF558 were prepared for use in complementation studies. Introduction of the recA allele slows the growth rate of these strains but does not affect their resistance to colicin A or tetracycline. After transfer of F'106 from strain CY99 to strains JF561 and JF562, several pyrD+ merodiploids were picked and purified through two single-colony isolations on selective medium. Four merodiploid isolates from each strain were tested for the presence of F'106 by their sensitivity to the male-specific phage f2 and their ability to transfer pyrD+ into the pyrD recA strain JF560. Strains that were

VOL. 128, 1976

THE tolF LOCUS

sensitive to the male-specific phage f2 and able to donate pyrD + were considered to be merodiploids. All eight strains picked and tested were merodiploids, and all eight were fully sensitive to colicin A and tetracycline. The tolF and cmlB alleles are both recessive. cmlB tolF merodiploid. Hfr strain JF404-4a was mixed with strains JF561 (recA pyrD toiF) and JF562 (recA pyrD cmiB), and pyrD+ colonies were selected on medium containing streptomycin. Under these conditions, only a few pyrD+ colonies appeared. Each colony probably carried an F' episome of unknown size but containing the point of origin of strain HfrH (13). Two colonies from each mating were chosen, and the presence of the episome was confirmed by the sensitivity to phage f2 and ability to donate pyrD+ to strain JF560 as described

607

above. The merodiploids prepared in this way were tested for sensitivity to colicin A and tetracycline. All of four strains tested were resistant to both agents. Since the episome in each case presumably carried the tolF allele as well as pyrD+, the phenotype of the merodiploids prepared from strain JF562 having the genotype pyrD cmlBIF' pyrD+ tolF demonstrated the lack of complementation between cmlB and tolF. This indicated that the tolF and cmlB mutations were in the same gene.

DISCUSSION The mutations in strain JF404-4a (tolF) and strain RE107 (cmlB) affect the same gene. This conclusion is based on similar positions on the genetic map, the low frequency of recombination between tolF and cmlB, identity of phenotype, and absence of genetic complementation. aro A tol F fab A Intragenic recombination frequencies have pdx C (cmI B) pyr D tol G t been measured, for example within the trpA 80 54(26) 57(54) 56 76 gene, and correlated with amino acid substitu8.9 54 tion in the gene product, the a subunit of tryp~~~8.0 tophan synthetase (12). Comparison of the fre55 36 quency of recombination among mutants in the trpA gene with the frequency of recombination FIG. 1. Position of tolF (cmlB) on the genetic map observed between tolF and cmlB suggests that ofEscherichia coli. The figure is an adaptation of the the mutations in the tolF and cmlB strains are portion corresponding to 20 to 21.5 min on the genetic separated by only a few base pairs on the chromap of Bachmann et al. -0

10-

(1). The numbers above the

arrows represent cotransduction frequencies, and the arrow heads indicate the unselected marker. Where reciprocal transductions were done (double arrow head), the cotransd uction frequencies were averaged. The data are from Tables 2 and 3 except for pdxC-

aroA (4) and pyrD-tolG-fabA (10). The numbers in parentheses are from Reeve and Doherty (16) for aroA-cmlB and pyrD-cmlB.

mosome.

The relationship among tolF, cmrB, and tolD loci was investigated since the phenotypes have features in common. Each mutation leads to both colicin tolerance and resistance to antibiotics. Clearly, the tolD locus is distinct both genetically and phenotypically from the tolF and cmlB loci. The tolD mutation is not geneti-

TABLE 5. Phenotypic comparison of isogenic tolF and cmlB strains Sensitivity to:a Colicinb

Other agents Chlor-

Strain A B D El E2 E3 la K L L-JF246 Ia M S1 DyesF Detergentsd

JF556 (wild type) JF557 (toiF) JF558

am-

pheni-

PolyTetracy- Oxyttcline racyc- myxin ln

S SS S S S S S S

S

S SS

R

R

Cole S

T S S S pT T S T T

T

S S S

R

R

R

R

R

R

T S S S pT T S T T

T

S S S

R

R

R

R

R

R

S

S

S

(cmiB) a Symbols: S, sensitive; T, tolerant; pT, partially tolerant; R, resistant. Strains JF404-4a and RE107 were shown to be

tolerant as previously described (10). b Colicin concentrations were all 200 U/ml except for colicin Ia, which was 80 U/ml. Colicin units and strains producing colicins A, El, E2, E3, Ia, and K have been previously described (10). Other colicin-producing strains were: AG097 (B), CA23 (D), 398 (L), 32T19F (M), P1 (31). c Dyes: 400 ug of eosine yellow/ml; 100 Ag of acridine orange/ml; 100 ;Mg of methylene blue/ml. d Detergents: 1% sodium dodecyl sulfate; 1% sodium deoxycholate; 0.1% Triton X-100. e Antibiotic sensitivity determined by the disk sensitivity test.

608

FOULDS

cally linked by transduction to either aroA or pyrD and produces a distinct pattern of tolerance to various colicins (2). Still, the question remains as to why mutations in separate loci should have similar pleiotropic effects. The pleiotropic effects of these mutations, colicin tolerance and increased resistance to antibiotics, may be explained by viewing the outer membrane of E. coli both as the initial permeability barrier of the cell and the primary site of interaction of colicins with the cells. Alterations in the composition or arrangement of outer membrane components (primarily phospholipids, protein, and lipopolysaccharide) could produce pleiotropic effects such as have been described here for tolF, cmlB, and tolD strains. For example, increased resistance to antibiotics may be due to decreased permeability of the outer membrane to these agents. Presumably the alteration in the membrane that results in decreased permeability may also change the sensitivity of the cell to colicins. Although I do not wish to suggest that all colicin-tolerant mutants have an altered outer membrane, at least some do. tolG mutants lack a major outer membrane protein (3). This is easily demonstrated by high-resolution polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (3). Similar treatment of materials prepared from tolF strains shows a marked difference in protein composition of the outer membrane when compared with material prepared from the wild-type strain. tolF and cmlB strains have much less of outer membrane protein 1 [nomenclature of Schnaitman (18)] (Foulds and Chai, manuscript in preparation). At present, the inability to transfer the tolD mutation by transduction cannot be explained. Since Hfr strain Gllel (tolD) can transfer colicin tolerance by conjugation, perhaps this strain contains an additional genetic alteration required for expression of the tolD phenotype. If so, locating the tolD locus by gradient mating techniques (2) would result in an apparent decreased inheritance of the tolD gene, placing it further away from the origin of transfer than it actually is. LITERATURE CITED 1. Bachmann, B. J., K. B. Low, and A. L. Taylor. 1976. Recalibrated linkage map of Escherichia coli K-12. Bacteriol. Rev. 40:46-167.

J. BACTERIOL. 2. Burman, L. G., and K. Nordstrom. 1971. Colicin tolerance induced by ampicillin or mutation to ampicillin resistance in a strain of Escherichia coli K-12. J. Bacteriol. 106:1-13. 3. Chai, T., and J. Foulds. 1974. Demonstration of a missing outer membrane protein in tolG mutants ofEscherichia coli. J. Mol. Biol. 85:465-474. 4. Cronan, J. E., Jr., D. F. Silbert, and D. L. Wulff. 1972. Mapping of the fabA locus for unsaturated fatty acid biosynthesis in Escherichia coli. J. Bacteriol. 112:206-211. 5. Davies, J. K., and P. Reeves. 1975. Genetics of resistance to colicins in E. coli K-12: cross-resistance among colicins of group A. J. Bacteriol. 123:102-117. 6. DeHaan, P. G., W. P. M. Hoekstra, C. Verhoef, and H. S. Felix. 1969. Recombination in E. coli. HI. Mapping by the gradient of transmission. Mutat. Res. 8:505512. 7. Eriksson-Grennberg, K. G., H. G. Bowman, J. A. T. Janson, and S. Thoren. 1965. Resistance of E. coli to penicillins. I. Genetic study of some ampicillin-resistant mutants. J. Bacteriol. 90:54-62. 8. Foulds, J. 1972. Purification and partial characterization of a bacteriocin from Serratia marcescens. J. Bacteriol. 110:1001-1009. 9. Foulds, J. 1974. Chromosomal location of the tolG locus for tolerance to bacteriocin JF246 in Escherichia coli K-12. J. Bacteriol. 117:1354-1355. 10. Foulds, J., and C. Barrett. 1973. Characterization of Escherichia coli mutants tolerant to bacteriocin JF246: two new classes of tolerant mutants. J. Bacteriol. 116:885-892. 11. Fredericq, P. 1965. A note on the classification of colicins. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. I Orig. 196:140-142. 12. Helinski, D. R., and C. Yanofsky. 1962. Correspondence between genetic data and the position of amino acid alteration in a protein. Proc. Natl. Acad. Sci. U.S.A. 48:173-183. 13. Low, B. 1968. Formation of merodiploids in matings with a class of rec- recipient strains of E. coli K-12. Proc. Natl. Acad. Sci. U.S.A. 60:160-167. 14. Reeve, E. C. 1966. Characteristics of some single step mutants to chloramphenicol resistance in E. coli K-12 and their interaction with R-factor genes. Genet. Res. 7:281-286. 15. Reeve, E. C. 1968. Genetic analysis of some mutants causing resistance to tetracycline in E. coli K-12. Genet. Res. 11:303-309. 16. Reeve, E. C. R., and P. Doherty. 1968. Linkage relationship of two genes causing partial resistance to chloramphenicol in Escherichia coli. J. Bacteriol. 96:1450-1451. 17. Rossi, J. J., and C. M. Berg. 1971. Differential recovery of auxotrophs after penicillin enrichment in Escherichia coli. J. Bacteriol. 106:297-300. 18. Schnaitman, C. A. 1974. Outer membrane proteins of Escherichia coli. HII. Evidence that the major protein of Escherichia coli 0111 outer membranes consists of four distinct polypeptides species. J. Bacteriol. 118:442-453. 19. Signer, E. R. 1966. Interaction of prophages at the att 80 site with the chromosome of E. coli. J. Mol. Biol. 15:243-255. 20. Stacey, K. A., and E. Simson. 1965. Improved method for the isolation of thymine-requiring mutants of Escherichia coli. J. Bacteriol. 90:554-555.

TolF locus in Escherichia coli: chromosomal location and relationship to loci cmlB and tolD.

Vol. 128, No. 2 Printed in U.S.A. JOURNAL OF BACTERIOLOGY, Nov. 1976, P. 604-608 Copyright © 1976 American Society for Microbiology tolF Locus in Es...
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